The detergent-soluble form of acetylcholinesterase was purified from the electric organ of the electric rays Narke japonica and Torpedo californica, and its properties were examined. The electric organ of N. japonica and T. californica contains three types of acetylcholinesterase: low-salt-soluble, asymmetric or tailed, and detergent-soluble forms. Results showed that in N. japonica, asymmetric forms were predominant, whereas in T. californica the detergent-soluble form was predominant. Low-salt-soluble acetylcholinesterase constituted 10% of the total acetylcholinesterase in both species. Detergent-soluble acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. Triton X-100 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted quantitatively by lowering the pH to 2.8. This simple procedure gave good yields. The purified enzymes gave single peaks at 6 S on sucrose gradients in the presence of detergent and polydisperse aggregates in the absence of detergent. Reduction of disulfide bonds gave peaks at 4.4 S. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the purified acetylcholinesterases gave bands with Mr of about 130 000 in the unreduced state and with Mr of 66 000 in addition to a very faint band of Mr 130 000 in the reduced state. The Mr-66 000 polypeptides were labeled with diisopropylfluorophosphate. Thus, the detergent-soluble acetylcholinesterases exist as dimers of the Mr-66 000 components. Two-dimensional electrophoresis of the purified enzymes indicated their homogeneity. The isoelectric points of both enzymes were 5.1 under the conditions employed. The two enzymes had very similar amino acid compositions, and contained more than 14% of neutral sugars and glucosamine. Monoclonal antibodies were raised to detergent-soluble acetylcholinesterase by the hybridoma technique; eight were obtained. All of them recognized the catalytic subunits of detergent-soluble and asymmetric acetylcholinesterase, and reacted only with detergent-soluble acetylcholinesterase in immunoblots. Four of the monoclonal antibodies inhibited the activities of both the detergent-soluble and asymmetric forms of acetylcholinesterase.